A critical function for CD4+CD25+ regulatory T (TR) in maintaining tolerance to allergens has been revealed by the observation that mutations in the forkhead factor Foxp3, which result in TR cell deficiency, lead to a syndrome of autoimmunity associated with the loss of oral tolerance, and severe food allergy. In parallel, emerging evidence has implicated TR cells in oral tolerance induction, and suggested a role for the commensal flora in promoting TR cell function. Accordingly, this proposal employs murine genetic models to examine the role in oral tolerance of the two populations of TR cells: the natural of thymically derived (nTR cells) and the induced TR cells (iTR), derived from conventional cells in the periphery by the action of TGF-[unreadable], and the mechanisms by which microbial flora promotes TR cell function. We hypothesize that 1) nTR and iTR cell populations mediate distinct functions in oral tolerance and 2) tolerance maintenance involves direct signaling by the commensal flora in TR cells via toll-like receptors (TLR). To address these hypotheses, we will make use of a number of informative genetic mouse models that we have derived including Foxp3-deficient, inducible and reporter mice. We propose the dissect the functions of nTR and iTR cells in oral hypersensitivity to food allergens using rescue models of Foxp3-deficeint mice that allow tracking of the respective cell populations in vivo. We also propose to employ Foxp3-deficient, TLR-deficient mice to examine the consequences of inactivating TLR signaling in TR cells on oral tolerance. The proposed studies will contribute to the elucidation of mechanisms by which TR cells maintain oral tolerance to allergens and how their breakdown promotes food allergy. CD4+CD25+ regulatory T (TR) cells express the forkhead transcription factor Foxp3 and are essential for the maintenance of mucosal tolerance, especially tolerance to innocuous food- antigens. Failure of TR cell function due to mutations in Foxp3 results in a syndrome of autoimmunity, allergic dysregulation and severe food allergy. The latter is frequently the cardinal clinical manifestation in milder forms of the disease, underscoring the essential contribution of TR cells to the control of allergic hypersensitivity to food. There are two types of TR cells, the so called 'natural'TR (nTR) cells that arise as a specific lineage in the thymus, and the 'induced'TR (iTR) cells are generated from conventional T cells in the periphery. The separate contributions of the iTR and nTR compartments to oral tolerance remain critical and unanswered questions. The proposed studies will test the hypothesis that complete tolerance requires both iTR and nTR cells. We postulate that each cell type serves a non-redundant role in the maintenance of oral tolerance, mandated by their distinct ontogeny and functional attributes. We also postulate that both populations dynamically respond to signals by the commensal microbial flora that promotes their in vivo functions in oral tolerance maintenance. The proposed studies would provide fundamental insights into the role of TR cell in tolerance maintenance to food allergens and mechanisms of food hypersensitivity pursuant to their deficiency.